Skeletal muscle wasting that occurs during aging and numerous other pathological conditions including HIV infection and cancer is a serious public health concern and was a financial burden of more than $18.5 billion to the U.S. healthcare system in 20001. Currently there are no effective strategies to promote muscle regeneration in aged, injured, diseased or physical inactive skeletal muscle. The inherent capacity of muscle tissue to regenerate itself is mediated by resident skeletal muscle stem cells (MuSCs)2,3,4. Recent advances by our laboratory and others have identified strategies to prospectively isolate MuSCs from murine skeletal muscle5,6,7,8,9,10,11. Due to the relatively nascent nature of the MuSC field, we are only just beginning to understand the mechanisms underlying MuSC regulation. Elucidating MuSC regulation will be critical to harness the potential of this adult, tissue-specific stem cell and lend to the development of therapeutics to overcome muscle atrophy. In recent studies we demonstrated that substrate elasticity, a physical property of the MuSC niche, is a potent regulator of MuSC viability and self-renewal12,13. Using a biomaterials approach in conjunction with in vivo functional assays, we showed for the first time that 'stemness'is maintained by culturing MuSCs on substrates that match the softness of muscle tissue. In this Career Development Proposal, we aim to extend this finding to gain further insight into MuSC regulation by physical features of the in vivo niche. Specifically, we will use a multidisciplinary approach to explore MuSC fate modulation by substrate rigidity and niche architecture and will focus on revealing mechanistic insights. In Aim 1, we will elucidate the mechanisms of MuSC regulation imposed by 2D substrate elasticity. This aim will test the hypothesis that MuSCs possess 'rigidity sensing'systems and that these systems transduce information about the physical environment to modify gene expression and ultimately, cell fate. The goal of Aim 2 is to engineer a skeletal muscle tissue mimic using engineered extra-cellular matrix (eECM) to interrogate MuSC physical regulation in the context of a 3D environment. We hypothesize that MuSC regulation by substrate rigidity will be fundamentally different in a 3D setting as compared to our 2D observations and aim to decouple the effects of 'dimensionality'on MuSC viability, proliferation and fate (quiescence, activation, self-renewal, differentiation). Finally, in Aim 3 we will investigate the role of 3D niche architecture on MuSC fate. We will use microfabricated models to test the hypothesis that MuSC quiescence and activation are determined by 3D niche architecture. The knowledge gained from these Aims will lend to our fundamental understanding of niche composition and MuSC regulation. The ultimate goal of this work is to potentiate development of cell-based or systemically delivered therapeutics designed to promote skeletal muscle regeneration in specific contexts of muscle wasting. If funded, this Career Development Award (CDA) would afford me the opportunity to expand my background in skeletal muscle biology, gain necessary knowledge of biomaterials approaches, achieve the capacity to interpret bioinformatics data and acquire expertise in microscopy;skills critical to my future success as an independent researcher (see timeline). Formal and informal interactions with my Advisory Committee will assess my progress on the proposed Aims and provide critique and advice. Finally, professional skills (e.g. mentoring, research presentation, time management, writing, etc) vital to my long term success as an academic will be gained during the K99 mentored period through Stanford courses designed to prepare postdocs for the career transition and through career development advice acquired by my advisory committee and my sponsor, Dr. Helen Blau. Together, the proposed studies and career development training will ensure I achieve my long term career goal;to establish a successful and independently-funded laboratory at a major University and use bioengineering approaches to study the molecular mechanisms that control muscle stem cells in the resting tissue and during regeneration specifically in the context of a 3D tissue. PUBLIC HEALTH RELEVANCE: Skeletal muscle is critical to our day to day movement and loss of muscle mass and/or function due to genetic disorders, aging, cancer or physical inactivity impairs quality of life and increases the risk of mobility related accidents. Adult skeletal muscle contains stem cells that are responsible for the day to day repair of skeletal muscle injury. The research in this proposal used an interdisciplinary approach and is focused on identifying the mechanisms regulating muscle stem cells in the tissue and aims to harness this knowledge to develop novel strategies for promote skeletal muscle repair in patients.